The fungus Candida albicans is the 4th most common cause of hospital-acquired bloodstream infections (BSI), and is a major cause of intravenous catheter-associated infections, urinary infections, skin and mucosal infections, and invasive disease in our veteran patient population. Despite advancements in medical care, the high mortality rate due to invasive Candida infections is no better than two decades ago. Thus, our ability to prevent, diagnose, and treat invasive Candida infections is still in need of great improvement. Although C. albicans is a normal human colonizer, it has the ability to cause disease through various specialized attributes. These virulence-associated factors include secretion of degradative enzymes that assist in tissue invasion, formation of elongated hyphal structures in a process termed filamentation, and establishment of complex structures called biofilms, which protect this fungus from antimicrobial drugs and host immune defenses. In previous studies, we examined the role of the pre-vacuolar secretory pathway in the secretion of virulence-associated proteins and biofilm formation in C. albicans, regulated by the vacuolar protein secretion genes VPS1, VPS4, and PEP12. We demonstrated that this pre-vacuolar secretory pathway contributes to secretion of a key degradative enzyme, secreted aspartyl protease, and is involved in aspects of filamentation, biofilm formation, and virulence. For example, we discovered that the C. albicans pep12 null mutant formed a biofilm that dramatically fragmented with minimal disturbance, and was defective in virulence in vivo. Expanding upon these studies, we next studied the late stages of secretion by examining key final steps in exocytosis regulated by the exocyst protein complex. In this work, we demonstrated that the exocyst-related SNARE proteins Sso2p and Sec9p were essential for viability in C. albicans, and were required for the secretion of aspartyl proteases and lipases, and hyphal formation. In contrast, several major components of the exocyst complex, including Exo70p and Exo84p, and the regulatory protein Sro77p did not appear to be required for filamentation. Next, we have begun studies of another essential component of the secretory pathway, that is, endocytosis and the endocytic pathway. This highly regulated, sequential pathway is involved in intake of extracellular materials and recycling of plasma membrane proteins and other components of the secretory pathway. Our main objective is to determine the role of endocytosis in secretion, filamentation, biofilm formation, and virulence. Further, we will study the specific contributions of endocytosis to filamentation from a mechanistic standpoint. This project will therefore examine the key hypotheses that: (i) specific genes in key steps of endocytosis are required for filamentation and biofilm formation, (ii) mutations in endocytosis pathway genes at key steps will result in attenuated virulence in vitro and in vivo, and (iii) C. albicans has novel genes and pathways that are involved in endocytosis. We will first study the specific functions of C. albicans END3, SYP1, PAL1, ENT1, and YAP1801, which are predicted to be involved in key early steps in endocytosis. We will also study RHO1 to determine if there is a novel route of endocytosis that exists in C. albicans, as described in Saccharomyces. Next, we will study a set of eight novel genes lacking human or Saccharomyces counterparts, involved in endocytosis or secretion, which we identified using a computational/comparative genomics approach. Then we will seek to identify novel genes involved in endocytosis by screening several C. albicans mutant library collections. All of these studies will be accomplished using genetic, biochemical, molecular and cell biology approaches. Expected Results: We expect to determine the functional role of specific genes related to key stages of endocytosis in in C. albicans, and define their link to filamentation and virulence in order to understand molecular mechanisms of pathogenesis and identify novel drug targets for further intervention.